CN110878298B - rRNA capture probe and application thereof - Google Patents

Abstract

The embodiment of the invention discloses an rRNA capture probe and an application method thereof. Wherein the rRNA capture probe is selected from one or more of the following three groups of probe sequences: the 5SrRNA probe sequence set includes: SEQ ID No.1 and SEQ ID No.2; the 16SrRNA probe sequence set includes: SEQ ID No.3 to SEQ ID No.24; the 23SrRNA probe sequence set includes: SEQ ID No.25 to SEQ ID No.76. Based on the capture probe, more than 90% of ribosomal RNA sequences in transcription products can be effectively removed, the technical problem that prokaryotic mRNA does not contain polyA tail and cannot be enriched and separated through oligo dT is solved, and sequencing data quantity and cost are greatly saved.

Description

rRNA capture probe and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to an rRNA capture probe and application thereof.

Background

Ribosomal RNA (rRNA) is the RNA type with the highest intracellular content, and accounts for more than 80% of the total amount of all RNA in the cell. It binds to proteins to form ribosomes, which transport specific amino acids under the sequence guidance of mRNA to synthesize protein peptide chains.

In prokaryotes, ribosomal RNAs are largely classified into three categories, 5S rRNA (about 120 nt), 16S rRNA (about 1540 nt) and 23S rRNA (about 2900 nt), and eukaryotes are largely classified into 5S rRNA (about 120 nt), 5.8S rRNA (about 160 nt), 18S rRNA (about 1900 nt) and 28S (about 4700 nt) rRNA.

Because of extremely high content of the ratio of the ribosomal RNA, the expression abundance, modification state and the like of other functional RNAs are researched in the sequencing of the total RNA, and a great amount of available data is invaded by the ribosomal RNA, so that how to effectively remove the ribosomal RNA for different species or accurately separate target type RNA for corresponding detection before detection becomes a core technical problem of early preparation of a sample in the research of 'transcriptomics' and 'apparent transcriptomics'.

In the process of implementing the present invention, the inventors found that the related art has the following problems: in eukaryotes, total mRNA containing poly A tail is usually fished using oligo dT magnetic beads and then analyzed for expression of cellular or tissue mRNA by high throughput sequencing through library construction.

But the mRNA content of prokaryotes is extremely low, about 2% -5% of the total RNA. Furthermore, most prokaryotic mRNAs do not have a poly (A) structure at the 3' end. Thus, it is very difficult to isolate and detect the mRNA of a prokaryote.

Disclosure of Invention

Aiming at the technical problems, the embodiment of the invention provides an rRNA capture probe and application thereof, so as to solve one or more problems in the existing rRNA removal method.

A first aspect of embodiments of the invention provides a rRNA capture probe for cyanobacteria. Wherein, the rRNA capture probes are prepared by mixing three groups of rRNA capture probes; wherein, the 5SrRNA probe sequence group consists of SEQ ID No.1 and SEQ ID No.2; the 16SrRNA probe sequence group consists of SEQ ID No.3 to SEQ ID No.24; the 23SrRNA probe sequence group consists of SEQ ID No.25 to SEQ ID No.76, and the length of each rRNA capture probe ranges from 30bp to 120bp; the length spacing between adjacent rRNA capture probes is less than 30bp.

Optionally, the rRNA capture probe further comprises a biotin label.

A second aspect of embodiments of the invention provides a whole transcriptome sequencing assay. Wherein the method comprises capturing and removing rRNA in the total RNA of the sample using an rRNA capture probe as described above.

In a third aspect of embodiments of the invention, a high throughput detection method for RNA modification is provided. Wherein the method comprises capturing and removing rRNA in the total RNA of the sample using an rRNA capture probe as described above.

Alternatively, the molar concentration of each rRNA capture probe is 0.1uM-1uM; the input amount of the total RNA of the sample is 50ug-10ng, and the concentration is more than or equal to 5ng/uL.

Alternatively, the molar concentration of each of the rRNA capture probes is 0.5uM.

Optionally, capturing and removing rRNA in the total RNA of the sample by the rRNA capture probe specifically includes:

hybridizing the rRNA capture probe with the sample total RNA in a hybridization buffer system to form a DNA-RNA hybrid strand;

after hybridization, rRNA in the DNA-RNA hybrid strand is specifically digested under the action of ribonuclease H;

the rRNA capture probe is specifically digested under the action of DNase I.

Optionally, capturing and removing rRNA in the total RNA of the sample by the rRNA capture probe specifically includes:

hybridizing the rRNA capture probe with the sample total RNA in a hybridization buffer system to form a DNA-RNA hybrid strand;

after hybridization, the DNA-RNA hybrid strand formed after hybridization is removed under selective degradation of double-strand specific nucleases.

Optionally, capturing and removing rRNA in the total RNA of the sample by the mixed rRNA capture probe, specifically including:

hybridizing the rRNA capture probe with the sample total RNA in a hybridization buffer system to form a DNA-RNA hybrid strand; wherein the rRNA capture probe is labeled with biotin;

the DNA-RNA hybrid strand formed after hybridization is removed from the total sample RNA by biotin-streptomycin affinity capture.

In the technical scheme provided by the embodiment of the invention, the specific rRNA capture probe is designed, so that more than 90% of ribosome RNA sequences in transcription products can be effectively removed based on the specific rRNA capture probe, the technical problem that prokaryote mRNA does not contain polyA tail and cannot be enriched and separated through oligo dT is solved, and sequencing data quantity and cost are greatly saved.

Since the rRNA capture probe captures only the rRNA sequence. Thus, when used in high throughput sequencing such as transcriptome sequencing analysis, non-coding RNA, lncRNA, tRNA, circle RNA, small RNA, and the like can be detected in addition to mRNA.

In addition, in RNA modification detection, other RNA modification conditions except for mammal rRNA can be detected only by a trace amount of total RNA (about 2 ug), and the method can be further used for analyzing modification levels of other species such as plants, microorganisms and the like, and has good application prospects.

Drawings

FIG. 1 is a schematic diagram of one embodiment of a whole transcriptome sequencing assay according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of an embodiment of a high throughput detection method for RNA modification according to an embodiment of the present invention.

SEQ ID No.1 and SEQ ID No.2 are capture probes designed for 5 SrRNA;

SEQ ID No.3 through SEQ ID No.24 are capture probes designed for 16 SrRNA;

SEQ ID No.25 to SEQ ID No.76 are capture probes designed for 23 SrRNA.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.

It will be understood that when an element is referred to as being "fixed" to another element, it can be directly on the other element or one or more intervening elements may be present therebetween. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or one or more intervening elements may be present therebetween. The terms "vertical," "horizontal," "left," "right," "upper," "lower," "inner," "outer," "bottom," and the like as used in this specification are based on the orientation or positional relationship shown in the drawings, merely to facilitate description of the invention and to simplify the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the invention. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used in this specification includes any and all combinations of one or more of the associated listed items. In addition, the technical features of the different embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The embodiment of the invention provides an rRNA capture probe. The rRNA capture probe takes 5S,16S and 23SrRNA sequences of cyanobacteria as reference sequences, and is designed according to the base complementary pairing principle to obtain three groups of probes, wherein the three groups of probes can respectively specifically combine 5S,16S and 23SrRNA with rRNA to form RNA-DNA hybrid double chains.

In the design process, the length range of each probe is controlled to be between 30bp and 120bp, the CG content in the probes is balanced, and the length interval between adjacent rRNA capture probes is smaller than 30bp.

Wherein the 5SrRNA probe sequence group comprises: SEQ ID No.1 and SEQ ID No.2; the 16SrRNA probe sequence set includes: SEQ ID No.3 to SEQ ID No.24; the 23SrRNA probe sequence set includes: SEQ ID No.25 to SEQ ID No.76.

In this example, a cyanobacterial rRNA capture probe is provided. Based on the same inventive concepts, one skilled in the art can also specifically design rRNA capture probes for other different species for use in gene sequencing or analysis of other organisms.

The rRNA capture probe provided by the embodiment of the invention can be suitable for various high-throughput sequencing platforms, such as illumine, huada MGI-seq or life, and the like, is applied to detection ranges of transcriptomics research, apparent transcriptomics research and the like, and is used for effectively removing rRNA in total RNA.

After the completion of the hybridization of the rRNA capture probe to the total RNA, the RNA-DNA hybrid duplex can be removed from the total RNA by any suitable means for subsequent sequencing and detection steps.

In the actual use process, each rRNA capture probe (i.e. different sequences in the sequence listing) can be adjusted to a set molar concentration and mixed, and then used as a complete rRNA capture probe set for hybridization with total RNA of a sample.

Specifically, the molar concentration of each rRNA capture probe can be controlled within the range of 0.1uM-1uM, and correspondingly, the input amount of the total RNA of the sample is 50ug-10ng, and the concentration of the RNA is ensured to be more than or equal to 5ng/uL.

Surprisingly, it was found that optimal hybridization was achieved with a molar concentration of 0.5. Mu.M for each rRNA capture probe and a total sample RNA input of between 500ng-2ug, and rRNA was removed from the total sample RNA.

In some embodiments, cleavage can be performed with ribonuclease H (RNase H) and deoxyribonuclease I (DNase I) to specifically remove probes and rRNA. The specific removal process may include the steps of:

first, the rRNA capture probes are hybridized with total sample RNA in a hybridization buffer system to form DNA-RNA hybrid strands.

Then, after hybridization is completed, rRNA in the DNA-RNA hybrid strand is digested specifically by ribonuclease H.

Finally, the rRNA capture probe is specifically digested under the action of deoxyribonuclease I, so that the purpose of removing rRNA in total RNA is achieved.

In other embodiments, double strand specific nucleases (DSNs) may also be used to the same effect. That is, after hybridization, the DNA-RNA hybrid strand formed after hybridization is removed under selective degradation of double-strand specific nuclease, thereby achieving the purpose of removing rRNA in total RNA.

In still other embodiments, a biotin-streptomycin affinity reaction may be used to capture DNA-RNA hybrid strands from total RNA. Of course, in this embodiment, the rRNA capture probe also includes a biotin label.

Thus, the hybrid strand of DNA-RNA formed after hybridization will be labeled with biotin, while the other RNA will not be labeled with biotin. Thus, the DNA-RNA hybrid strand will be captured by the streptomycin by an affinity reaction, thereby removing the DNA-RNA hybrid strand from the total RNA.

FIG. 1 is a flow chart of a method for whole transcriptome sequencing assay according to an embodiment of the present invention. The whole transcriptome sequencing detection utilizes the rRNA capture probe to achieve the purpose of removing rRNA from total RNA in a sample. As shown in fig. 1, the method comprises the steps of:

s110: sample total RNA was prepared.

In particular, the total RNA sample may be extracted by any suitable method, such as the Trizol method or using commercially available kits. The total RNA of the extracted sample needs to be detected by an electrophoresis method, and the obvious main bands of 5S,16S and 23SrRNA are determined, so that the total RNA has no degradation and the concentration is not lower than 5ng/ul.

S120: the mixed rRNA capture probes are hybridized with the total RNA of the sample to form DNA-RNA hybrid strands to capture rRNA in the total RNA of the sample.

Specifically, 1-2ug of total RNA of the sample and mixed rRNA capture probes (the molar concentration of each rRNA capture probe is controlled at 0.5 uM) can be taken under a hybridization buffer system, denatured under preset hybridization reaction conditions (95-75 ℃, cooled to 20 ℃ at a speed gradient of 0.1-0.5 ℃ per second, and reacted for 5 minutes) for hybridization.

S130: DNA-RNA hybrid strands are removed from the total RNA of the sample.

Specifically, any of the methods provided in the examples above can be used to specifically remove the hybrid strand of DNA-RNA from the total RNA of the sample. Based on the inventive concepts disclosed in the above examples, a person skilled in the art may also adjust or change the specific method used, for example, to use other markers instead of biotin, according to the needs of the actual situation.

S140: the RNA was fragmented and reverse transcribed into cDNA.

Under the action of salt ions, the rest of the total RNA in the sample can be fragmented into a desired fragment length range of 150-300bp by heating. The fragmented RNA may be reverse transcribed into cDNA by reverse transcriptase and the second strand synthesis is performed by DNA polymerase.

S150: the cDNA was repaired and ligated to universal linker.

Specifically, the cDNA obtained after reverse transcription may be modified by adding A and phosphorylating with dNTPs as substrates under the action of enzymes such as Polymerase, phosphorylase, etc. (including but not limited to T4 DNA Polymerase, bst DNA Polymerase, klenow Fragment (3 '-5' exo-), PCR DNA Polymerase, and T4 Polynucleotide Kinase).

Then, the universal linker was ligated under the action of T4 DNA ligand. Specifically, universal adaptors can be ligated to cDNA using a double-stranded DNA blunt end ligation, a cohesive end such as T-A ligation, adaptor introduction of single-stranded DNA random primer extension Shen Jie guide, single-stranded DNA ligation, or the like.

The specific universal linker modifications used may also be set by the skilled artisan as desired in practice, including, but not limited to, hydroxy/methylation modifications, phosphorothioate modifications, LNA bases modifications, and the like.

S160: after PCR amplification, high throughput sequencing was performed.

PCR amplification can be performed by using the universal adaptor ligation product obtained in step S150 as an amplification template, with the aid of an artificially synthesized primer sequence. After the amplified product is purified, a quantitative PCR mode is selected, and after Agilent 2100 insert detection is carried out on the amplified target region/site library, high-throughput sequencing is carried out.

FIG. 2 shows a high throughput detection method for RNA modification according to an embodiment of the present invention. The RNA modification may be any suitable type of RNA modification, such as m6A or m5C, etc. (in this example, the m6A modification is described). The method has low requirement on the input amount of the total RNA of the sample, and can detect the total RNA of the sample by using a trace amount of the total RNA of the sample.

As shown in fig. 2, the RNA modified high throughput detection method may include the steps of:

s210: sample total RNA was prepared and fragmented.

In particular, the total RNA of the sample may be fragmented in any suitable manner, such that it is fragmented to a desired length in the range of 50bp to 300 bp. Specifically, RNA can be fragmented in a set buffer system at 70-95 ℃ for 1-10 minutes.

Surprisingly, it was found that the optimal length range for fragmentation is 100-250bp. That is, in the total RNA of the sample after fragmentation, the fragments of the RNA are distributed within the optimal length range.

S220: magnetic beads and antibody complexes were prepared.

In the actual use process, equal amounts of protein A/G coupled magnetic beads can be respectively taken and mixed, the volume of each magnetic bead is 5-50ul, and 100-200ul of immune enrichment buffer solution is used for washing the magnetic beads (150mM NaCl,10mM Tris-HCl pH7.5,0.1% IGEPAL CA-630) for 1-2 times.

Then, the beads were re-selected in 100-500ul of the immune enrichment buffer, and 5-20ug of anti-m6A antibody and 1ul of protease inhibitor (including Cocktail, PMSF protease inhibitor) were added and reacted at room temperature for 1-6h or spun overnight at 4 ℃.

S230: the m6A modified fragmented RNA was enriched by antibody immunization.

Based on the preparation of the magnetic bead-antibody complex in step 220, the supernatant may be first removed with a magnetic rack, and after washing the magnetic bead-antibody complex 2-3 times with 100-500ul of the immune enrichment buffer, the supernatant may be removed.

After the total RNA which is broken and purified is denatured by heating at 75 ℃ for 5-10 minutes and incubating on ice for 5 minutes, 2-5ug of fragmented total RNA is further mixed with the complex while 1-5ul of RNase inhibitor, 100ul of high concentration immune enrichment buffer (750mM NaCl,50mM Tris-HCl pH7.5, 0.5% IGEPAL CA-630) and fragmented RNA (total volume controlled at 500 ul) are added, and finally the mixture is spun at 4 ℃ for 2-4 hours.

S240: and (3) separating and purifying the m6A modified fragmented RNA.

Prior to isolation, the immune-enriched reaction mixture obtained in step 230 may be washed twice with immune-enriched buffer (50mM NaCl,10mM Tris-HCl pH7.5,0.1% IGEPAL CA-630) and twice with high-salt buffer (500mM NaCl,10mM Tris-HCl pH7.5,0.1% IGEPAL CA-630), respectively. The non-modified RNA fragments are then removed using a suitable purification procedure and the fragments are recovered to contain m6A modified total RNA (including mRNA, small RNA, tRNA, lncRNA and the vast majority of rRNA).

Specifically, purification can be performed by any suitable method (e.g., RNeasy Mini Kit of Qiagen) using any suitable m6A competition elution or Kit purification.

S250: the mixed rRNA capture probe is hybridized with the m6A modified fragmented RNA to form a DNA-RNA hybrid strand to capture rRNA in the m6A modified fragmented RNA.

Specifically, m6A modified fragmented RNA and mixed rRNA capture probes (the molar concentration of each rRNA capture probe is controlled to be 0.1-0.5 uM) can be taken under a hybridization buffer system, and hybridized under preset hybridization reaction conditions (denaturation at 95-75 ℃, gradient cooling to 20 ℃ at a speed of 0.1-0.5 ℃ per second and reaction for 5 minutes).

S260: removing the DNA-RNA hybrid strand.

Specifically, any method provided in the above examples can be selected to specifically remove the hybrid strand of DNA-RNA, thereby achieving the effect of rRNA removal. Where the capture probe includes a biotin label, the rRNA can be removed by incubating with M280 streptomycin magnetic beads, and fishing the DNA-RNA hybrid strand.

Based on the inventive concepts disclosed in the above examples, a person skilled in the art may also adjust or change the specific method used, for example, to use other markers instead of biotin, according to the needs of the actual situation.

S270: and (3) recovering m6A modified fragmented RNA after rRNA removal, and carrying out reverse transcription, PCR amplification and library construction sequencing.

After step S260, an m6A modified RNA fragment from which rRNA was removed can be recovered. dNTP (10 uM) and N6 primer can be added for reaction at 70-75 ℃ for 2-5min, the mixture is put on ice for denaturation, TSO primer, reverse transcriptase, RNase inhibitor and reverse transcription buffer solution are added into the reaction system, and the mixture is uniformly mixed for reaction at 42 ℃ for reverse transcription and terminal transferase reaction.

After the reverse transcription reaction, PCR Primer1 and PCR Primer2 were added to the reaction system, respectively, to carry out PCR amplification. The finally obtained amplified product is purified by magnetic beads, and library fragments and molar concentration are detected by using an Agilent biological analyzer 2100 and QPCR and subjected to high-throughput sequencing to construct a library.

The following describes in detail the specific application procedure of the rRNA capture probe provided by the embodiment of the present invention with reference to specific examples.

Example 1: prokaryotic transcription component library sequencing analysis of cyanobacteria

1.1 System6803 Pool Mix (i.e., a mixture of multiple rRNA capture probes shown in the gene sequence listing) with total RNA:

first, a preparation Hybridization Buffer:500mM Tris-HCl (pH 7.0), 1M NaCl. Cyanobacteria total transcriptome RNA 200ng was taken in a 0.2ml PCR tube and Hybridization Buffer 3ul,Probe Mix 0.5ul was added in a total volume of 15ul. The reaction was carried out on a PCR apparatus at 95℃for 2min, followed by a gradient cooling (0.1 ℃ C./sec) to 22℃for 5min. Immediately after the reaction, the mixture is placed on an ice box, so that RNA degradation is reduced as much as possible.

1.2 RNase H enzyme digestion):

in the PCR tube of the previous reaction, nuclease free water 1ul,RNase H Buffer 2ul,RNase H (5U/ul) was 2ul and the total volume was 20ul. The reaction was carried out on a PCR instrument at 37℃and a hot cover temperature of 47℃for 30min. Immediately after the reaction, the mixture is placed on an ice box, so that RNA degradation is reduced as much as possible.

1.3 DNase I enzyme digestion:

in the PCR tube of the previous reaction Nuclease free water 4.5.5 ul,10 XDNase I Buffer 3ul, DNase I (2U/ul) 2.5ul, total volume of 30ul. The reaction was carried out on a PCR instrument at 37℃and a hot cover temperature of 47℃for 30min. After the reaction, it was purified by 1.8X RNA clean XP beads and eluted into 10ul Nuclease free water.

1.4 RNA fragmentation):

in the last step, 5X First Strand Buffer ul of eluent is added, the mixture is reacted for 10min at 94 ℃ in a PCR instrument, and the mixture is immediately placed on an ice box after the reaction is finished.

1.5 RNA reverse transcription):

wherein, synthesis of reverse transcription first strand: n6 primer (0.1 ug/ul) 0.5ul was added to the RNA fragmented in the above step, reacted at 65℃for 5min in a PCR instrument, cooled on an ice box, and RNase Inhibitor (40U/ul) 0.5ul and dNTP (10 mM) 0.5ul,100mM DTT 0.5ul were added to the RNA fragment in a total volume of 15ul. The reaction was carried out at room temperature for 2min, superscript II (200U/ul) was added to the mixture, and the reaction was carried out in a PCR apparatus according to the following procedure:

Step 1：25℃，10min；Step 2：42℃，40min；Step 3：70℃，15min；Step4：4℃，Hold。

synthesis of reverse transcribed second strand: a total of 15.25ul of the synthesized one-stranded cDNA was transferred into a 1.5ml EP tube, and ddH2O 20ul, 5X Second Strand Buffer 10ul, dNTP (10 mM) 2ul, RNase H (5U/ul) 0.5ul, DNA pol I (10U/ul) 2.5ul was added to the tube in a total volume of 50ul. Mixing, placing on a thermo mixer, reacting at 16 ℃ for 2h, intermittently vibrating at 350rpm for 15s, and standing for 2min.

Purifying: adding 1.8Xegene beads (magnetic beads need to be balanced for 30min at room temperature in advance) into the cDNA, mixing uniformly, standing for reaction for 5min, placing the mixture on MPC for 2min, and carefully sucking out the supernatant; adding 200ul of 80% ethanol, washing the magnetic beads twice, removing the ethanol as clean as possible for the last time, adding 30ul of EB solution to resuspend the magnetic beads after drying residual ethanol, reacting for 5min at room temperature, placing the magnetic beads on MPC for 2min, and sucking the supernatant into a new 1.5ml EP tube for the next reaction.

1.6 cDNA end repair:

to 30ul of cDNA product obtained in the previous reverse transcription, 5ul of 10 XPNK buffer,1ul of dNTP (10 mM), 1.2ul T4 DNA polymerase (3U/ul), 0.2ul Klenow Fragment,1.2ul T4 Polynucleoyide Kinase,11.4ul H2O, and total volume of 50ul were added. Mixing, and placing on a Thermomixer for reaction for 30min at 20 ℃.

Purifying: adding 1.2 Xegene beads (magnetic beads need to be balanced for 30min at room temperature in advance) into the product of the previous step, mixing uniformly, standing for reaction for 5min, placing the mixture on MPC for 2min, and carefully sucking out the supernatant; adding 200ul of 75% ethanol, washing the magnetic beads twice, removing the ethanol as clean as possible for the last time, adding 20ul of EB solution to resuspend the magnetic beads after drying residual ethanol, reacting for 5min at room temperature, placing the magnetic beads on MPC for 2min, and sucking the supernatant into a new 1.5ml EP tube for the next reaction.

1.7 End addition a):

to 20ul of post-end repair product, 2.3ul of 10 Xblue buffer,0.5ul of dATP (5 mM), 0.5ul of Klenow (3 '-5' exo), and a total volume of 23ul were added. Mixing, and placing on a thermo mixer for reaction for 30min at 37 ℃.

1.8 Universal joint connection):

to 23ul of product after addition of the sticky end to the end of the previous step, 1.2ul 2X Rapid ligation buffer,1.2ul T4 DNA ligase,1ul PEI adapter (2 uM), 0.1ul 10mM ATP, total volume 26.5ul was added. Mixing, and placing on a Thermomixer for reaction for 20min at 20 ℃.

Purifying: adding 1.8Xegene beads (magnetic beads need to be balanced for 30min at room temperature in advance) into the product of the previous step, mixing uniformly, standing for reaction for 5min, placing the mixture on MPC for 2min, and carefully sucking out the supernatant; adding 200ul of 75% ethanol, washing the magnetic beads twice, removing the ethanol as clean as possible for the last time, adding 20ul of EB solution to resuspend the magnetic beads after drying residual ethanol, reacting for 5min at room temperature, placing the magnetic beads on MPC for 2min, and sucking the supernatant into a new PCR tube for the next reaction.

1.9 PCR amplification and product purification:

to 20ul of the product with universal linker attached, 10ul of 5 Xkapa HiFi buffer,0.8ul kapa HiFi polymerase,2ul 10mM dNTP,1.5ul 10uM universal primer,1.5ul 10uM index primer,14.2ul H2O total volume 50ul was added. After mixing, the mixture was placed on PCR and reacted according to the following procedure:

cycling for 1 time at 95 ℃ for 2 min; cycling for 12 times at 94 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 45 s; cycling for 1 time at 72 ℃ for 3 min; preserving heat at 12 ℃.

Purifying: adding 0.9Xegene beads (magnetic beads need to be balanced for 30min at room temperature in advance) into the product of the previous step, mixing uniformly, standing for reaction for 5min, placing on MPC for 2min, and carefully sucking out the supernatant; adding 200ul of 75% ethanol, washing the magnetic beads twice, removing the ethanol as clean as possible for the last time, adding 20ul of EB solution to resuspend the magnetic beads after drying residual ethanol, reacting for 5min at room temperature, placing the magnetic beads on MPC for 2min, and sucking the supernatant into a new 1.5ml EP tube.

1.10 High throughput sequencing data statistics):

the secondary Gao Tongli Lang sequencing is carried out after the quality control of the library, the data comparison shows that the rRNA removal effect is good, the machine-on data comparison rate is reliable, the residual rRNA removal rate is about 7%, the experimental requirements are met, and the result is similar to that of a similar commercial probe (human/mouse).

1.11 Off-machine data processing statistics:

the statistical results are shown in the following table, which shows higher genome alignment and lower rRNA alignment.

Sample name	C1
		Original off-line data volume	4983065700
Raw data Q20	96.62％
		Raw data Q30	91.72％
Effective data volume (Gb)	4.417
		Effective data yield	99.92％
Alignment data	28062897
		Comparison rate	84.55％
rRNA residual Rate	7.09％

Example 2: trace RNA methylation detection based on probe removal of ribosomal rRNA:

2.1 Magnetic bead-antibody complex preparation:

30ul of protein A magnetic beads and 30ul of protein G magnetic beads, respectively, are added to a 1.5ml centrifuge tube, washed twice with 200ul of immune enrichment buffer, respectively, and the supernatant is removed; 500ul of immune enrichment buffer, 5ug of anti-m6A antibody, was added to a centrifuge tube containing magnetic beads and the reaction was spun overnight at 4 ℃.

2.2 Antibody enrichment m6A total RNA fragment:

taking 5ug of total RNA to carry out fragmentation reaction in a 20ul system, reacting for 5min at 70 ℃, adding 2ul of EDTA to terminate the reaction after the reaction, and recovering the fragmented RNA by an ethanol precipitation method, wherein the length of the fragmented RNA is 100-200bp.

Placing the magnetic bead antibody complex reacted overnight on a magnetic frame to remove the supernatant, respectively washing the magnetic bead-antibody complex with 300ul of immune buffer solution for 2 times, removing the supernatant by the magnetic frame, finally adding 5ul of RNase inhibitor into the washed magnetic bead-antibody complex, recovering and purifying RNA, and rotating and reacting for 2 hours at 4 ℃ under an enrichment buffer system in a total volume of 500 ul.

2.3 M6A modified RNA isolation from total RNA:

the magnetic bead-antibody-RNA complexes were washed 2 times with 1ml of each of the immunization buffer, the low-salt immunization buffer and the high-salt immunization buffer, and the capture complexes were purified by adding 200ul of RLT buffer to the commercial Kit RAeasy Mini Kit (Qiagen).

2.4 Removal of enriched rRNA based on capture probes:

hybridizing the enriched RNA with 1ul of synthetic probe System6803 Pool (a mixture of a plurality of rRNA capture probes shown in a gene sequence table), denaturing at 75 ℃ for 5min, cooling to 20 ℃ at a gradient of 0.1-0.5 ℃/s, reacting for 5min, adding 2ul of RNase H into a reaction product, reacting for 30min at 37 ℃, adding 2.5ul of DNase I into a reaction System, reacting for 30min at 37 ℃, and purifying by using 1.8X magnetic beads after the reaction.

2.5 Enriched RNA library construction:

to the bead-RNA mixture, 2ul of N6 primer and 2ul of dNTP and 4ul of ultra-pure water were added, reacted at 72℃for 3min, and immediately placed on ice for 5min. After centrifugation, 1ul of reverse transcriptase, 0.5ul of RNase inhibitor, 4ul of one-strand synthesis buffer,1ul of DTT,4ul of 5M betaine, 0.12ul of 1M magnesium chloride and 0.2ul of TSO primer were added and reacted on a mix PCR apparatus according to the following steps:

the reaction is carried out for 90min at 42 ℃ for 1 time, for 2min at 50 ℃ for 2min, for 2min at 42 ℃ for 2 times, and for 15min at 70 ℃ for 1 time.

After the reverse transcription reaction, 25ul of PCR reaction mixture, 1ul of primer1 (10 uM) and 1ul of primer2 (10 uM) and 3ul of ultrapure water were further added to the reaction system, PCR amplification was performed for 10 to 14 cycles, and gene sequencing was performed based on the PCR amplification products, to construct a corresponding RNA library.

In summary, the embodiment of the invention designs the ribosome removing probe by analyzing the characteristics of the transcription product and rRNA sequence of cyanobacteria, builds an RNA-DNA hybridization elimination system and an RNA library building process, and can effectively remove more than 90% of ribosomal RNA sequences in the transcription product.

For transcriptome sequencing analysis, the rRNA capture probe can effectively overcome the technical problem that prokaryote mRNA does not contain polyA tail and cannot be enriched and separated through oligo dT, and greatly saves sequencing data quantity and cost.

In addition, since probes capture only rRNA sequences, non-coding RNAs, lncRNA, tRNA, circle RNA, and small RNAs, etc., can be detected in addition to mRNA expression, modification, by high throughput sequencing, as compared to eukaryotic mRNA fishing.

In the application of RNA modification detection, the method flow of the invention not only can design a synthetic probe sequence and detect other RNA modification conditions outside mammal rRNA in trace total RNA (about 2 ug), but also can be further used for analyzing the modification level of other species such as plants, microorganisms and the like and realizing another RNA modification detection technology and kit.

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Sequence listing

<110> Shenzhen City Yi Gene technology Co., ltd

<120> rRNA capture probes and uses thereof

<141> 2019-11-13

<160> 76

<170> SIPOSequenceListing 1.0

<210> 1

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 1

cggactattg tgccgtgggg caacccccag agtatcgtcg ccgctgtcac 50

<210> 2

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 2

cacaaccgag tgcgggatgg gatcggagtg gttccatgac gctaaagaca 50

<210> 3

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 3

gagttgcagc ctgcaatctg aactgaggcc gggtttgatg ggattcgctt 50

<210> 4

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 4

cgagctcgct gcccgttgtc ccgaccattg tagtacgtgt gtagcccaag 50

<210> 5

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 5

ggggcatgat gacttgacgt catccccacc ttcctccggt ttgtcaccgg 50

<210> 6

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 6

ctctagagtg cccaacttaa tgatggcaac taaaaacgag ggttgcgctc 50

<210> 7

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 7

ggacttaacc caacatctca cgacacgagc tgacgacagc catgcaccac 50

<210> 8

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 8

cctggctccc taaggcactc ccacgtttcc gcaggattcc agggatgtca 50

<210> 9

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 9

ggtaaggttc ttcgcgttgc atcgaattaa accacatact ccaccgcttg 50

<210> 10

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 10

ccccgtcaat tcctttgagt ttcacacttg cgtgcgtact ccccaggcgg 50

<210> 12

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 12

taacgcgtta gcttcggcac ggctcgggtc gatacaagcc acgcctagta 50

<210> 12

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 12

gtttacggct aggactacag gggtatctaa tccctttcgc taccctagct 50

<210> 13

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 13

cctcagtgtc agtttcagcc cagtagcacg ctttcgccac cgatgttctt 50

<210> 14

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 14

atctacgcat ttcaccgcta cactgggaat tcctgctacc cctactgtac 50

<210> 15

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 15

cttgcagttt ccaccgctcc tatggagtta agctccattc tttaacggca 50

<210> 16

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 16

cataaccacc tacggacgct ttacgcccaa taattccgga taacgcttgc 50

<210> 17

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 17

cgtattaccg cggctgctgg cacggagtta gccgatgctt attcatcagg 50

<210> 18

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 18

cagaacttct tccctgataa aagaggttta caatccaagg accttcctcc 50

<210> 19

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 19

cggtattgct ccgtcaggct ttcgcccatt gcggaaaatt ccccactgct 50

<210> 20

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 20

cgtaggagtc tgggccgtgt ctcagtccca gtgtggctgc tcatcctctc 50

<210> 21

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 21

gctactgatc gttgccatgg taggctctta ccccaccatc tagctaatca 50

<210> 22

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 22

gcccatcttc agacgataaa tctttcacct ttcggcacat tgggtattag 50

<210> 23

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 23

ttccaactgt tgtccccatt ctgaaggtag gttctcacgt gttactcacc 50

<210> 24

<211> 45

<212> DNA

<213> Cyanobacteria

<400> 24

cgtccgccac taagttccga agaactccgt tcgacttgca tgtgt 45

<210> 25

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 25

gtcaagccct cggtctatta gtactcctcg acttcatcca ttactggact 50

<210> 26

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 26

ctagagccta tcaacgggta gtcttcccgt gaccttactg gcttaacacc 50

<210> 27

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 27

gtactcatct tgaggtgggc ttcccactta gatgctttca gcggttatcc 50

<210> 28

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 28

cacttggcta ccctgcgttt accgttggca cgataacagg tacaccagag 50

<210> 29

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 29

acttcccggt cctctcgtac taaggaagtc tcctctcaat actcttacgc 50

<210> 30

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 30

cggatatgga ccgaactgtc tcacgacgtt ctgaacccag ctcacgtacc 50

<210> 31

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 31

atgggcgaac agcccaaccc ttgggacgta ctaccgcccc aggttgcgat 50

<210> 32

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 32

acatcgaggt gccaaacctc cgcgtcgatg tgaactcttg gcggagatca 50

<210> 33

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 33

tatccctaga gtaactttta tccgttgagc gacggccctt ccactcagtg 50

<210> 34

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 34

gatcactaaa gccgactttc gtccctgttt gacttgtcag tctcacagtc 50

<210> 35

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 35

ccttctgctt ttacactctt cggctgattt ccaaccagcc tgaggaaacc 50

<210> 36

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 36

cgcctccgtt accttttagg aggcgaccgc cccagtcaaa ctgcccacct 50

<210> 37

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 37

gtccttctcc cggataacgg gaacaagtta gaattctagc ctcaccagag 50

<210> 38

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 38

ctcaccgttg actccattac tcccacaaga gcaacttcat agtctcccac 50

<210> 39

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 39

tgcgcaagca aagcccgaac ccaattccaa gctacagtaa agcttcatag 50

<210> 40

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 40

tctgtccagg tgcagggagt ccgtatcttc acagacaatc ctatttcgcc 50

<210> 41

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 41

ctctccgaga cagtgcccag atcgttacgc ctttcgtgcg ggtcggaact 50

<210> 42

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 42

acaaggaatt tcgctacctt aggaccgtta tagttacggc cgccgttcac 50

<210> 43

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 43

cttcagtcgc tagcttcagg attactccct gaccaacttc cttaaccttc 50

<210> 44

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 44

tgggcaggcg tcagccccca tactgcgtct tacgactttg cggagacctg 50

<210> 45

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 45

tggtaaacag tcgcctgggc ctattcactg cgacctccat tgctggaggc 50

<210> 46

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 46

tctcccgaag ttacggggta attttgccga gttccttaga gagagttacc 50

<210> 47

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 47

cccttagtat tctctacctt cctacctgtg tcggtttcgg gtacaggtga 50

<210> 48

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 48

attaacgtgg ttcgggcttt tcttggaagc ttgacatcat gcacttcgtc 50

<210> 49

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 49

gagactcccc atcacacctc tgctcaagac gttttcgccg tctctcatcg 50

<210> 50

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 50

atgcttggac tggtaaccaa cttccagttg cactagcctt ctccgtcccc 50

<210> 51

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 51

caatctataa tcagtaccgg aatattgacc ggttgtccat cgactacgcc 50

<210> 52

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 52

cctcgcctta ggtcctgact aaccctccgc ggacgagcct tccggaggaa 50

<210> 53

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 53

ggatttcggg gtatgggatt ctcacccata ttttcgctac tcaagccgac 50

<210> 54

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 54

acttctgtac tgtccacacc tgcttgccgc tagtgcttca ccctatacag 50

<210> 55

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 55

cccctaccac tcatatctga gtccacagct tcggtacatc acttagcccc 50

<210> 56

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 56

tttcggcgca ggagcgcttg accagtgagc tattacgcac tcttttaagg 50

<210> 57

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 57

gcttctaggc aaacctcctg gttgtcaatg cactcccacc tcctttctca 50

<210> 58

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 58

gatgatttgg ggaccttagc tggtggtctg ggctgtttcc ctttcgacga 50

<210> 59

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 59

ttatccccca ccgtcttact ggtcgtttct tcttgggtat tctgagtttg 50

<210> 60

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 60

tttggtacag ctctcgccgc ccgcagcgaa acagtgcttt accccccaag 50

<210> 61

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 61

ttacaaccgc tgcgcctaaa cacatttcgg ggagaaccag ctagctccgg 50

<210> 62

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 62

ttggcatttc acccctaacc acagctcatc cgctaatttt tcaacattag 50

<210> 63

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 63

cggacctcca cttagtgtta cctaagcttc atcctggcca tggttagatc 50

<210> 64

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 64

gttcgggtct acaaattgtg actaacgccc tattcaggct cgctttcact 50

<210> 65

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 65

tcggtgcaac acaccttaac ctgccacaac ctgtaagtcg ccggctcatt 50

<210> 66

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 66

caggcacacg gtcactcgtt taatcgagct cccattgctt gtaggctaac 50

<210> 67

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 67

catgttctat ttcactcccc tcaacggggt tcttttcacc tttccctcgc 50

<210> 68

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 68

ttacgctatc ggtcacacag tagtatttag ccttaccggg tggtcccggc 50

<210> 69

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 69

aatcggaatt ccacgagctc cgacctactc gggatacagc taggctgatt 50

<210> 70

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 70

ttcgactaca ggactttcac ctcctctggt gcagttttca gctgcttcgt 50

<210> 71

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 71

tactcagtcc acgttgctgt cccacgaccc caatcttcga aaagattggt 50

<210> 72

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 72

tgttccccgt tcgctcgccg ctacttggag aatcactttt gttttctttt 50

<210> 73

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 73

gctactaaga tgtttcagtt cactaggttt gctctctccc gcctatttta 50

<210> 74

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 74

ggtagttcac ttgggttgcc ccattcggac acctccggat caatgcttgt 50

<210> 75

<211> 50

<212> DNA

<213> Cyanobacteria

<400> 75

ccagctcccc gaagcgtttc gtcggtaacc acgtccttct tcgcctctgt 50

<210> 76

<211> 39

<212> DNA

<213> Cyanobacteria

<400> 76

gtgccaaggt atccaccgtt agccctttgt agcttgacc 39

Claims (6)

1. A cyanobacteria rRNA capture probe, which is characterized by being prepared by mixing three groups of rRNA capture probes; wherein, the liquid crystal display device comprises a liquid crystal display device,

the 5SrRNA probe sequence group consists of SEQ ID No.1 and SEQ ID No.2;

the 16SrRNA probe sequence group consists of SEQ ID No.3 to SEQ ID No.24;

the 23SrRNA probe sequence group consists of SEQ ID No.25 to SEQ ID No. 76;

the length of each rRNA capture probe ranges from 30bp to 120bp; the length spacing between adjacent rRNA capture probes is less than 30bp.

2. The rRNA capture probe of claim 1, further comprising a biotin label.

3. A whole transcriptome sequencing assay method, wherein rRNA in total RNA of a sample is captured and removed using the rRNA capture probe of claim 1.

4. A method according to claim 3, wherein the molar concentration of each rRNA capture probe is 0.1uM to 1uM; the input amount of the total RNA of the sample is 50ug-10ng, and the concentration is more than or equal to 5ng/uL.

5. The method of claim 4, wherein the molar concentration of each of the rRNA capture probes is 0.5uM.

6. The method of claim 3, wherein the rRNA capture probe is used to capture and remove rRNA from total RNA of the sample, comprising:

hybridizing the rRNA capture probe with the sample total RNA in a hybridization buffer system to form a DNA-RNA hybrid strand;

after hybridization, rRNA in the DNA-RNA hybrid strand is specifically digested under the action of ribonuclease H;

the rRNA capture probe is specifically digested under the action of DNase I.